1. Field of the Invention
The physical separation of semen into purified X- and Y-chromosome bearing sperm populations using flow cytometry and sorting has proven to be effective in humans, cattle, swine, sheep and rabbits for gender preselection.
The success of the sorting process is dependent on the accuracy and efficiency of analyzing sperm for DNA content. High resolution flow cytometric DNA analysis of sperm is hampered by its uneven emission of fluorescence. Due to the sperm head's flat shape, compactness of chromatin and a high index of refraction, fluorescence from the edge is much brighter than from the flat side of the sperm (Gledhill et al., J Cell Physiol. 87:367-376 (1976); Johnson et al., Cytometry 7:268-273 (1986); and Pinkel et al., Cytometry 3:1-9 (1982)]. This fact, coupled with random orientation, results in a broad fluorescence distribution hiding specific subpopulations with different contents of DNA within a sample. A solution for this problem is to use an epiillumination flow cytometer employing co-axial flow characteristics. However, this system is generally not suitable for cell sorting. The only solution that incorporates DNA analysis and sorting is to collect only fluorescence from properly oriented cells using an orthogonal flow cytometer/cell sorter (Johnson et al. and Pinkel et al., supra). In the orthogonal system, a 90.degree. fluorescence detector is used for detection of properly oriented sperm. A bright signal indicates that a sperm's bright edge is effectively orientated to the 90.degree. detector and consequently the flat side will face the laser beam. A forward fluorescence detector (0.degree.) is added to the cell sorter to collect the fluorescence from the flat face opposite to the laser beam. The forward fluorescence signal when collected from the flat side of the properly orientated sperm is an accurate measurement of DNA content (Johnson et al. and Pinkel et al., supra).
Improved high efficiency sorting can only be valuable for sperm sexing if three criteria are met:
1) It does not decrease the accuracy of the DNA measurement (maintain 90% purities). PA1 2) Stability of sort is not compromised (time not wasted adjusting cell sorter). PA1 3) Enough sperm can be recovered enabling their use for in vitro fertilization and artificial insemination.
This invention relates to a novel nozzle which is designed to orient and sort a large fraction of sperm independent of sheath fluid velocity and sample rate.
2. Description of the Prior Art
The orientation of sperm (cells) is random in conventional cell sorters. For sperm sexing, cell sorters have been modified by replacing the sample injection needle with a beveled needle [Dean et al., Biophys. J. 23:7-13 (1978); Fulwyer, J. Histochem. Cytochem. 25:781-783 (1977); Johnson et al., supra; Pinkel et al., supra; Stovel, et al., Biophys. J. 23:1-5 (1978); and Welch et al., Cytometry 17(Suppl. 7): 74 (1994)]. This beveled needle forces a larger proportion of sperm to pass the laser beam in the proper orientation because it reshapes the cylindrical sample stream into a thin ribbon. The beveled needle helps to orient sperm, especially sperm heads, e.g., sperm without their tail. The sample core leaving the beveled needle will be in the shape of a ribbon, which applies orienting forces to the sperm. However this ribbon only exists when the sample stream is narrow; that is, under low sample pressure and concomitant low sample rate. These conditions are not advantageous for efficient sperm sorting. Also, the improvement in orientation attributed to use of a beveled needle is less pronounced for living and motile sperm. Only 20-40% of intact viable sperm are correctly orientated using this system [Johnson, Reprod. Fertil. Dev. 7:893-903 (1995)]. This means that between 60% and 80% of the detected sperm are not analyzed for DNA analysis. The ability to sort intact and viable cells in conjunction with improved orientation efficiency would lead to a much higher sorting efficiency.
Kachel et al. [J. Histochem. Cytochem. 25:774-780 (1977)] disclose an asymmetric Plexiglas.RTM. chamber for orienting flat particles (fixed chicken erythrocytes) on a microscope. Kachel et al. suggest that the simplest flow path for applying the necessary hydrodynamic focusing forces consists of a tube with an elliptical cross-section and ending in an elliptical outlet having a long axis at right angles to the long axis in the cross-section of the constricting elliptical tube. This device was not proposed for use in combination with a cell sorter.